We have made significant progress during the prior funding period in assessing the role of FLT3 mutations in AMI, and the development of murine models to test FLT3 inhibitors. These have included analysis of FLT3-ITD expression alone, and in combination with cooperating alleles such as PML-RARalpha. Working with Project 1, Project 2 has been instrumental in preclinical development of FLT3 inhibitors by showing efficacy in murine models of disease, resulting in Phase I and Phase II trials of two different FLT3 inhibitors in Project 5. We plan to move forward with additional studies of the role of FLT3-ITD in leukemogenesis using conditional knock-in alleles. In Specific Aim 1, we will analyze the in vivo activity of FLT3-ITD and activation loop alleles, and try to understand the relative predilection of FLT3-ITD for myeloid lineage disease, and of FLT3 activation loop alleles for lymphoid disease. We will use high speed multiparameter flow cytometry to test the hypothesis that these alleles have differential effect on cell fate determination at the multipotent progenitor stage (LMPP/MPP) where FLT3 is highly expressed during hematopoietic development. We will study the mechanism whereby FLT3-ITD, in contrast with FLT3 WT, is a potent activator of STATS using mutations in the context of FLT3 that abrogate this activity. In Specific Aim 2, we will explore cooperating effects of these accurate genotypic models of FLT3-ITD mediated disease through crosses with several complementing alleles including, C/EBPalpha, and MLL fusions, working with Projects 3 and 4. These in turn will serve as useful in vivo models for testing novel combination therapies that are developed in Project 1. We .will also pursue recent findings suggesting that retroviral FLT3-ITD retroviral integration sites may contribute to pathogenesis of AML in the Cathepsin G - PML-RARalpha model of cooperativity. In Specific Aim 3, we will develop murine models of myeloproliferative disease (MPD) mediated by the JAK2V617F allele that we and others have recently identified.. Finally, we will use murine models of MPD as a platform for testing novel JAK2 inhibitors for development of clinical trials in Project 5. Overall, this is a highly interactive Project that will build on a proven track record of success and preclinical development of novel therapies for myeloid malignancies.